With ever-increasing applications of nanoscale materials in the biomedical field, the impact of nanoparticle size on cellular uptake efficiency, dynamics, and mechanism has attracted numerous interests but still leaves many open questions. A combined “multiphoton imaging-UV/Vis spectroscopic analysis” method was applied for the first time for quantitative visualization and evaluation of the cellular uptake process of different-sized (15-, 30-, 50-, and 80-nm) gold nanoparticles (AuNPs). Quantitative analysis of the size effect on cellular uptake behavior of AuNPs from a stack of three-dimensional multiphoton laser scanning microscopy images is obtained. The technique allows for differentiating AuNPs present in external and internal subcellular components, giving detailed information for elucidating cellular uptake dynamics without particle labeling. The data show that the internalization extent of AuNPs is highly dependent on particles’ sizes and incubation time. Due to sedimentation, 50- and 80-nm AuNPs are taken up to a greater extent than 15- and 30-nm particles after exposure for 24 h. However, the smaller particles’ uptake velocity is significantly faster in the first 10 h, indicating a disparity in uptake kinetics for different-sized AuNPs. The finding from this study will improve our understanding of the cellular uptake mechanisms of different-sized nanoparticles and has great implications in developing AuNP-based drug carriers with various sizes for different purposes.